Nucleic acid free ghosts preparations

ABSTRACT

The invention relates to preparations of bacterial ghosts which are substantially free of living bacterial cells and/or nucleic acids and their use in pharmaceutical preparations.

DESCRIPTION

[0001] The invention relates to preparations of bacterial ghosts whichare substantially free of living bacterial cells and/or nucleic acidsand their use in pharmaceutical preparations.

[0002] Empty bacterial membranes, so-called bacterial ghosts, areprepared by controlled heterologous expression of a gene which effects apartial lysis of the cellular membrane of bacteria, particularlygram-negative bacteria (EP-A-0 291 021). For example, the lytic gene maybe the bacteriophage PhiX174 gene E encoding a polypeptide which isinserted into the cell membrane complex of gram-negative bacteria andleads to the formation of a transmembrane tunnel structure through theinner and outer membrane. The inner diameter of this tunnel structure isin the range of about 40 to 1,000 nm. The cytoplasmic components may beliberated by means of this tunnel structure, wherein an empty cellmembrane having an intact morphology, a so-called bacterial ghost, isobtained. The use of bacterial ghosts as dead vaccines or adjuvants andthe preparation of recombinant bacterial ghosts carrying heterologoussurface proteins in their membrane structures is disclosed in WO91/13555and WO93/01791.

[0003] Although the lytic process leading to an empty membrane withoutcytoplasmic structures is quite effective, a certain amount, usuallyabout one cell in 10⁴ cells remains intact. In order to render evensafer the use of bacterial ghosts as dead vaccines, particularly forapplications in human medicine, it is necessary to provide ghostpreparations, which contain a substantially lower number of livingbacterial cells.

[0004] Surprisingly, it was found that the efficiency of the lyticprocess for the preparation of bacterial ghosts may be increased byco-expression of the lytic gene together with a gene encoding an enzymewhich is present and hydrolytically active in the cytoplasm of the cell,wherein said enzyme is capable of hydrolyzing cytoplasmic compoundsnecessary for non-limited function of the cell, wherein the enzyme ispreferably selected from nucleases, phospholipases, lipases, lysozymes,proteases and carbohydrases. The expression of such enzymes is describedin EP-B-0 635 061. More preferably, the enzyme is a nuclease,particularly a truncated and/or mutated Staphylococcus aureus nucleasewhich is disclosed in WO95/10614.

[0005] Regulated co-expression of a bacterial lysis gene, e.g. thebacteriophage PhiX174 gene E and a nuclease gene, results in ansynergistic increase of efficiency of the lytic process andcorrespondingly in a substantial reduction of living bacterial cells ina ghost preparation.

[0006] Thus, a first aspect of the present invention relates to apreparation of bacterial ghosts, which is substantially free of livingbacterial cells. Preferably, the ratio of ghosts to living cells(determined as CFU by plating) is at least 10⁶:1, more preferably atleast 10⁷:1, still more preferably at least 10⁸:1, and most preferablyat least 10⁹: 1.

[0007] Further, the present invention relates to a preparation ofbacterial ghosts, which is substantially free of nucleic acids,particularly substantially free of nucleic acids having a length of ≧10nucleotides. Preferably, no nucleic acid is detected by Real Time-PCRhaving a lower detection limit of about 1-2 pg of DNA per 1×10⁶, inparticular per 2×10⁶ ghosts and/or living or dead bacteria per ml. TheReal Time-PCR is preferably carried out as described in Example 2 usingsuitable primers for a gene within the bacterial cell, e.g. anantibiotic resistance gene.

[0008] Further, the invention relates to a pharmaceutical compositioncomprising a preparation of bacterial ghosts as described above and apharmaceutically acceptable carrier, diluent and/or adjuvant. Thecomposition is suitable as a vaccine or an adjuvant, e.g. animmunostimulating compound, which is used together with an immunogenagainst which an immune-reaction shall be raised. The composition issuitable for use in human medicine and veterinary medicine. Moreover,the ghosts may be used as carriers for therapeutic and diagnosticagents.

[0009] Preferably, the ghosts are derived from gram-negative bacteria,which may be selected e.g. from Escherichia coli, Klebsiella,Salmonella, Pseudomonas, Vibrio, Actinobacillus, Haemophilus,Pasteurella, Bordetella and Helicobacter. Furthermore, the ghosts may berecombinant ghosts, i.e. ghosts carrying heterologous proteins, e.g.immunogens, in the membrane.

[0010] The ghosts may be administered according to known procedures,e.g. orally, intranasally, intraocularly, topically or parenterally.Depending on the mode of administration the composition may beformulated as an injectable or aerogenally applicable solution orsuspension, as an oral composition, e.g. as tablet, capsule or dragée,as cream or ointment. Furthermore, the composition may be formulated asa reconstitutable lyophilisate.

[0011] The ghost preparation of the invention may be prepared by amethod comprising the steps:

[0012] (a) providing bacterial cells comprising

[0013] (i) a gene encoding a lytic protein capable of forming a tunnelstructure in the bacterial membrane and

[0014] (ii) a gene encoding an enzyme capable of hydrolyzing cytoplasmiccomponents in the bacterial cells,

[0015] (b) optionally cultivating the bacterial cells under conditions,wherein the lytic gene and the enzyme gene are not expressed,

[0016] (c) subjecting the bacterial cells to conditions, wherein thelytic gene and the enzyme gene are expressed and the cytoplasmiccomponents of the bacterial cells are degraded and liberated and

[0017] (d) obtaining the resulting bacterial ghosts.

[0018] Preferably, the lytic gene and the enzyme gene are in operativelinkage with a regulatable expression control sequence. More preferably,the lytic gene and the enzyme gene are each in operative linkage with aseparate, usually different regulatable expression control sequence.

[0019] Thus, the expression of both genes may be initiated separately,e.g. at different times of the cultivation procedure.

[0020] In a particularly preferred embodiment, the cells are cultivatedunder repressing conditions for both the lytic gene and the enzyme gene.Then, the expression of the enzyme is induced, e.g. when the enzyme geneis under control of a chemically regulatable promoter such as the lacpromoter or a derivative thereof by adding an inducer, such as IPTG.

[0021] More preferably, the enzyme is expressed in a form which is atleast partially inactive and which may be activated at a later stage byaddition of a prosthetic group to the culture.

[0022] Then, subsequently, e.g. after 20 min up to 1.5 h, particularlypreferably after about 45 min, the expression of the lytic gene isinduced, e.g. when the lytic gene is in operative linkage with atemperature-regulatable promoter, such as the lambda PR or PL promoterby a temperature shift to 42° C. Then, after about 30 min up to 2 h,e.g. at about 90 min, the enzyme is activated by adding a prostheticgroup required for its function, e.g. metal ions, such as Mg²⁺ and/orCa²⁺.

[0023] Further, the invention relates to a bacterial cell comprising (i)a gene encoding a lytic protein capable of forming a tunnel structure ina bacterial membrane and (ii) a gene encoding an enzyme capable ofhydrolyzing cytoplasmic components in the bacterial cell. This cell maybe used as a starting material in a method for obtaining a preparationof bacterial ghosts, which is substantially free of living cells and/orwhich is substantially free of nucleic acids, particularly substantiallyfree of nucleic acids having a length of ≧10 nucleotides.

[0024] The lytic gene and/or the enzyme gene may be located on a vector,e.g. on the same vector or on different vectors. For example, the vectormay be an extra-chromosomal plasmid having an origin of replication anda selection marker gene.

[0025] It should be noted that the disclosure of all patent documentsrecited in the specification above is incorporated herein by reference.

EXAMPLES

[0026] 1. Preparation of Bacterial Ghosts using a Combination of E Lysisand Nuclease Treatment

[0027] 1.1 Material

[0028] The E. coli strain NM522 was used. The E. coli cells weretransformed with the plasmid pML1, which is a lysis plasmid carrying thePhiX174 gene E under control of a wild type lambda promoter and akanamycin resistance gene, or pSNUC1, which is a nuclease plasmidcarrying the snuc gene (Genbank V01281, J01785, M10924; D. Shortle, Gene22 (2-3), 181-189 (1983)) under control of a lac promoter and anampicillin resistance gene.

[0029] 1.2 Experimental Design

[0030] The nuclease of the gram-positive bacterium Staphylococcus aureusis an enzyme which hydrolyzes nucleic acids due to its exonuclease andendonuclease activity. Experiments showed the combined expression ofgene E and snuc increased the inactivation rate of ghosts and results ina reduced nucleic acid content.

[0031] 1.3 Two-Vector Systems

[0032] In these systems protein E and SNUC are encoded on two differentexpression plasmids. Actually two different systems were tested. lysisexpr. expr. sys- plas- of resi- SNUC of resi- tem mid gene E stanceorigin plasmid SNUC stance origin 1 pML1 cl857- kana- P15A pSNUC1 A1/O4/ampi- ColE1 pR mycin O3 cillin 2 pAW cl857- tetra- Col pHS cl857- kana-P15A 12 pR cyclin E1 SNUC pR mycin

[0033] Best results were achieved using system 1 and followingexperimental design:

[0034] Overnight cultures of E. coli NM522 carrying plasmids pML1 andpSNUC1 grown at 28° C. with shaking were diluted in LB medium containingkanamycin and ampicillin to an OD₆₀₀ about 0.05 to 0.08. Cultures weregrown at 28° C. with shaking to an OD₆₀₀ about 0.25 to 0.3 (time-45).Then IPTG was added in a final concentration of 5 mM to induceexpression of SNUC. 45 minutes later the cultures were shifted to 42° C.(time 0) to induce expression of gene E. 90 minutes after shift (time90) calcium ions and magnesium ions were added in final concentrationsof 10 mM and 1 mM, respectively, to activate the enzymatic activity ofSNUC.

[0035] OD₆₀₀ was measured at time-45 and after that in intervals of 30minutes starting at time 0 and CFU (colony forming units) weredetermined at the following times: −45, 0, 30, 60, 90, 120, 180, 240,300, 360, 420, 480, 540, 600 and 1320. These experiments resulted intotal inactivation of E. coli within 6 to 22 hours.

[0036] 2. Measurement of DNA Content in Bacterial Ghosts by RealTime-PCR

[0037] The residual DNA content was analyzed in the pellet as well as inthe supernatant by agarose gel. In order to quantify the DNA content theconcentration of kanamycin resistance gene and ampicillin resistancegene corresponding to the number of plasmids pSNUC1 and pML1,respectively, was measured by Real Time-PCR.

[0038] 2.1 Devices and Chemicals

[0039] Real Time Cycler: Corbett Research (distribution: genXpressService & Vertrieb GmbH, Vienna, Austria)

[0040] Polymerase: Dynazyme (Finzyme)

[0041] DNA-dye: SYBR-Green I 10,000×(Molecular Probes)

[0042] dNTPs: 10 mM each (Roche) 2.2 Mastermix for Real Time-PCR Amountin μl for 1 Amount in μl for 20 Components batch (25 μl) batches (500μl) Polymerase buffer 2.5 50  Polymerase 0.25 5 Primer 1 0.1 2 Primer 20.1 2 dNTPs (10 mM each) 0.5 10  SYBR l (10× in H₂O) 0.25 5 H₂O 20.8416  Template-DNA 0.5 (10)

[0043] 2.3 Programs for PCR (Kan/Amp) 94° C.  2 min 40 cycles: 94° C. 28sec 60° C. for Kan (62° C. for Amp)  1 min 72° C.  1 min  4° C. untilthe end fusion curve: 50° C.-96° C.

[0044] Primer: Kan-start: 5′-atgagccatattcaacgggaaa-3′ Kan-stop:5′-ttagaaaaactcatcgagcatca-3′ Amp-start: 5′-atgagtattcaacatttccgtgtc-3′Amp-stop: 5′-ttaccaatgcttaatcagtgagg-3′

[0045] 2.4 DNA-Templates

[0046] Plasmid Standards

[0047] The plasmids pSNUC1 and pML1 were prepared from overnightcultures (28° C./Amp or rather Kan) of E. coli NM522 via alkaline lysisand stored at 4° C. after RNase-treatment in TE-buffer. The absolute DNAamounts of the plasmid standards were determined fluorimetrically: thestandards were prepared from calf-thymus-DNA and after dyeing with theDNA-dye HOECHST 33342 (molecular probes) a calibrating plot wasprepared. The plasmid standards were also treated with the dye andanalyzed fluorometrically. From the calibration straight line theDNA-content in the plasmid samples could be determined.

[0048] pML1: 0.32 μg/μl

[0049] pSNUC1: 0.19 μg/μl

[0050] From these master-plasmid-standards the dilutions (up to 10⁻⁷)were produced in a TE-buffer. pSNUC1- pML1- Standard dilutionconcentration/μl concentration/μl original 190 ng 320 ng 10⁻¹ 19 ng 32ng 10⁻² 1.9 ng 3.2 ng 10⁻³ 190 pg 320 pg 10⁻⁴ 19 pg 32 pg 10⁻⁵ 1.9 pg3.2 pg 10⁻⁶ 190 fg 320 fg 10⁻⁷ 19 fg 32 fg

[0051] Samples from Lysed Cultures

[0052] Every time 1 ml culture was taken and centrifuged. 0.5 ml of thesupernatant were analyzed, the remainder thrown away.

[0053] The pellet (from 1 ml culture) was subjected to a total DNApreparation (Easy-DNA; invitrogen) and the preparation added to 100 μlTE+RNase.

[0054] The supernatant (from 0.5 ml culture) was two times extractedwith phenol/chloroform and precipitated with ethanol. The pellet wasadded to 50 μl TE+RNase.

[0055] In order to quantify the samples from lysed cultures the plasmidstandards 10⁻², 10⁻⁴ and 10⁻⁵, a zero control (without DNA template) anda number (10-12) of DNA samples were analyzed simultaneously. All of thesamples (including zero-control and standards) were analyzed in a doublebatch via real time-PCR.

[0056] Each time 0.5 μl of the DNA-preparations were employed astemplates.

[0057] After concentration values with regard to the genes to bequantified had been assigned to the plasmid standards, thequantification of the samples was performed completely automatically bythe PCR-apparatus. DNA-samples, which led to an increase in the productyield in the real-time kinetics only after the standard 10⁻⁵, orprovided no signal or one too weak, similar to the standards 10⁻⁶/10⁻⁷,were regarded as being “below the detection limit” when determining theconcentration.

[0058] 2.5 Real Time-PCR

[0059] The plasmid standards produced (dilutions 10⁻¹-10⁻⁷) wereanalyzed by means of real time-PCR, in order to determine the detectionlimits. After analysis of the data of the Real Time-PCR-measurements andthe fusion curves those dilutions of further analyses were excluded,which provided no signal or a signal too weak. Those fluorescence valueswere considered to be signals too “weak”, which were derived fromPCR-product-amounts that turned out to be smaller than the averageplateau value of the higher standards, whereby the “correct” product(Kan/Amp) provided the smaller portion of the fluorescence signal.

[0060] At both standard curves a linear measurement range within theconcentration range of 10⁻¹-10⁻⁵ could be found.

[0061] Detection limits: pML1: 1.67 pg (0.5 μl in dilution 10⁻⁵)

[0062] pSNUC1: 1 pg (0.5 μl in dilution 10⁻⁵)

3. FIGURES

[0063]FIG. 1 shows the lysis kinetics after expression of the lysis geneE in E. coli NM522 (pML1).

[0064]FIG. 2 shows the results of a real-time PCR (kanamycin resistancegene) in E. coli NM522 (pML1) cells.

[0065]FIG. 3 shows the lysis kinetics after co-expression of lysis geneE and staphylococcal nuclease (SNUC) in E. coli NM522 (pML1+pSNUC).

[0066]FIGS. 4a and 4 b show the results of a real-time PCR (kanamycinand ampicillin resistance gene) in E. coli NM522 (pML1+pSNUC).

[0067] From a comparison between FIGS. 1 and 2 (prior art) and the FIGS.3 and 4 (present invention) it can be seen that by the co-expression oflytic gene and nuclease gene a surprising reduction of the livingbacterial cells (expressed as CFU) and the nucleic acids (expressed asresult of the real-time PCR) is obtained.

[0068] From this comparison the advantages achieved by the presentinvention can be clearly gathered.

1 4 1 22 DNA Escherichia coli 1 atgagccata ttcaacggga aa 22 2 23 DNAEscherichia coli 2 ttagaaaaac tcatcgagca tca 23 3 24 DNA Escherichiacoli 3 atgagtattc aacatttccg tgtc 24 4 23 DNA Escherichia coli 4ttaccaatgc ttaatcagtg agg 23

1. A preparation of bacterial ghosts which is substantially free ofliving bacterial cells.
 2. The preparation of claim 1 wherein the ratioof ghosts to living cells is at least 10⁶:1.
 3. The preparation of claim2 wherein the ratio of ghosts to living cells is at least 10⁸:1.
 4. Thepreparation of bacterial ghosts which is substantially free of nucleicacids.
 5. A pharmaceutical composition comprising the preparation ofclaim 1 and a pharmaceutically acceptable carrier, diluent and/oradjuvant.
 6. The composition of claim 5 which is a vaccine.
 7. Thecomposition of claim 5 which is an adjuvant.
 8. The composition of claim5 for use in human medicine.
 9. The composition of claim 5 for use inveterinary medicine.
 10. A method for obtaining a preparation ofbacterial ghosts of claim 1 comprising the steps: (a) providingbacterial cells comprising (i) a gene encoding a lytic protein capableof forming a tunnel structure in the bacterial membranes and (ii) a geneencoding an enzyme capable of hydrolyzing cytoplasmic components in thebacterial cells (iii) a gene encoding an enzyme capable of hydrolyzingcytoplasmic components in the bacterial cells, (b) optionallycultivating the bacterial cells under conditions wherein the lytic geneand the enzyme gene are not expressed, (c) subjecting the bacterialcells to conditions wherein the lytic gene and the enzyme gene areexpressed and the cytoplasmic components of the bacterial cells aredegraded and liberated and (d) obtaining the resulting bacterial ghosts.11. The method of claim 10, wherein the gene encoding the lytic proteinis the bacteriophage phiX174 gene E or a lytically active fragmentthereof.
 12. The method of claim 10, wherein the gene encoding thehydrolytic enzyme is a nuclease gene.
 13. The method of claim 12,wherein the nuclease is a truncated and or mutated Staphylococcus aureusnuclease gene.
 14. The method of claim 10, wherein the lytic gene andthe enzyme gene are in the operative linkage with a regulatableexpression control sequence.
 15. The method of claim 14, wherein theylytic gene and the enzyme gene are each in operative linkage with aseparate regulatable expression control sequence.
 16. The method ofclaim 15, wherein the expression of the lytic gene and the expression ofthe enzyme are induced at different times.
 17. A bacterial cellcomprising (i) a gene encoding a lytic protein capable of forming atunnel structure in the bacterial membrane and (ii) a gene encoding anenzyme capable of hydrolyzing cytoplasmic components in the bacterialcell.
 18. The cell of claim 17, wherein the lytic gene and/or enzymegene are located on one or several vectors.